ExpiCHO™: Surpassing the Performance of 293 in a Transient ... · PDF fileability to...
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PEI Max ExpiFectamineCHO 0 1 2 3 hIgG Titer (g/L) (No Enhancer or Feed) (With Enhancer and Feed) 6.113 7.240 7.637 8.850 10.621 AU 0.000 0.010 0.020 0.030 0.040 0.050 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 6.816 7.232 7.633 8.848 10.651 AU 0.000 0.010 0.020 0.030 0.040 0.050 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 6.110 7.450 7.635 8.854 10.663 11.963 AU 0.000 0.010 0.020 0.030 0.040 0.050 Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Days of Culture hIgG Titer (g/L) 0 1 2 3 4 5 6 7 8 9 10 11 0.0 0.5 1.0 1.5 2.0 Control 125 mL Flask High Titer Protocol Chao Yan Liu, Virginia Spencer, Shyam Kumar, Jian Liu, Ping Liu, Sara Barnes, Henry Chiou*, Jonathan F. Zmuda. Thermo Fisher Scientific, 7335 Executive Way, Frederick MD, *5791 Van Allen Way, Carlsbad, CA. (A) ExpiCHO-S cells. (B) Stability of protein expression over 18 passages. (C) Growth curve for ExpiCHO-S cells grown in standard shake flask culture. Figure 6. ExpiCHO Protocol(s) ABSTRACT & INTRODUCTION CHO cells are the predominant host for biotherapeutic protein expression, with roughly 70% of licensed biologics manufactured in CHO. Multiple attributes make CHO cells desirable for bioproduction including the ability to adapt to high-density suspension culture in serum-free and chemically-defined media and the incorporation of post- translational modifications that are biologically-active in humans. For these reasons, the ability to produce transient CHO-derived proteins early on during drug development is highly advantageous to minimize, as much as possible, changes in protein quality/ function observed when moving from R&D to bioproduction. Unfortunately, CHO cells express lower levels of protein than HEK293 cells in existing transient systems, in some instances 50-100 times less than the best 293-based systems, and only modest titer improvements are obtained through the optimization of individual components of existing transient CHO workflows. To address the significant unmet need for higher transient CHO protein titers, systems-based approaches were employed whereby the latest advances in cell culture media, feeds, transfection reagents and expression enhancers were optimized in conjunction with a new high-expressing CHO cell clone to generate a simple and robust workflow for transient protein expression in CHO cells capable of generating gram per liter protein titers in 10-14 days. These advances will allow for unprecedented access to CHO-derived proteins early on during candidate selection and may serve to revolutionize the use of CHO cells for transient protein expression during the drug development process. CONCLUSIONS We describe a systems-based approach for enhancing levels of transient protein production in CHO cells that allows for the production of recombinant proteins at levels exceeding those of the Expi293 system while maintaining activity, purity and glycosylation patterns comparable to those observed in stably transfected CHO-S cells. This performance enhancement was made possible through the incorporation of multiple novel reagents including: (1) a high-expressing CHO cell clone, (2) a CD/AOF culture media that allows for high density CHO growth and transfection, (3) an optimized CHO cell transfection reagent, (4) a novel CHO feed optimized for transient transfection culture conditions, (5) a post-transfection enhancer solution and (6) a simple to perform workflow. ACKNOWLEDGEMENTS We would like to thank Michael Gillmeister for performing the glycan analysis on the human IgG samples and Brian Paszkiet for assisting with the transfection efficiency studies. ExpiCHO™: Surpassing the Performance of 293 in a Transient CHO Expression System Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com Figure 1. Systems-Based Approach to Increased Transient Protein Expression Figure 2. Workflow for Identifying High-Expressing CHO Clones CHO cells were transiently transfected and evaluated for protein expression using the ClonePix system (Molecular Devices). Selected clones were further evaluated via transfection with plasmids for multiple proteins. Control protein titers using an existing CHO clone as well as Control titers for the pre-clonal pool. ExpiCHO-S Cell Line Attributes • Derived from GMP CHO-S cells • Adapted for high-density culture • High specific productivity (~27pcd) • No clumping during expression runs • Short doubling time (~17 hours) • Stable growth and expression over 20+passages A B C Figure 3. Characterization of ExpiCHO-S Cells V. Workflow ExpiCHO Expression Media Attributes • No supplementation required • One medium for growth and transfection • Formulated specifically for transient transfection • Chemically-defined (CD) • Animal origin-free (AOF) • Serum-free • Protein-free • Manufactured under cGMP • Supports high-density cell growth • Matched to a specific feed • Free from regulatory/import/export limitations III. ExpiCHO Expression Medium and ExpiCHO Feed hIgG Titer (g/L) ExpiCHO Enhancer 0 1 2 3 4 _ + hIgG Titer (g/L) One Two 0 1 2 3 ExpiCHO Feed (A) Protein titers are reduced by 50% or more without the addition of the ExpiCHO Enhancer reagent. (B) Two equal volume feeds on Days 1 and 5 post-transfection double protein titers. Figure 4. Optimization of ExpiCHO Enhancer and Feed Addition IV. ExpiFectamineCHO Transfection Reagent (A) When used in conjunction with ExpiCHO Feed and Enhancer, ExpiFectamineCHO generates greater than 30-fold higher titers than PEI alone. (B) Despite the high density of cells at the time of transfection, plasmid DNA levels as low as 0.5 µg/mL of culture volume generate maximal protein titers, corresponding to half of the industry standard of 1.0 µg/mL plasmid DNA. Figure 5. Characteristics of ExpiFectamineCHO Transfection Reagent 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 hIgG Titer (g/L) DNA Concentration (μg/mL) 30 fold II. ExpiCHO Expression Medium and ExpiCHO Feed Viability Viable Cell Density Titer VI. Kinetics of Protein Production A B C A B (A) (Green line) Standard Protocol consisting of one feed and no temperature shift. (Blue line) High Titer Protocol consisting of one feed and temperature shift to 32ºC. (Red line) hIgG kinetics using the Max Titer Protocol consisting of two feeds and temperature shift to 32ºC. (B) Viability post transfection. (C) Viable cell density post transfection for Standard and Max Titer protocols. Figure 7. Kinetics of hIgG Expression, Viability and Viable Cell Density VII. System Flexibility: Ultra-High Density Transfection Ultra-high density transfection can generate protein titers in half the time. (A) (Red line) Transfection of ExpiCHO-S cells at density of 6x10 6 cells/mL using the Standard protocol. (Blue line) High density transfection of ExpiCHO-S cells at 10x10 6 cells/mL. (B) (Red line) Transfection of ExpiCHO cells at density of 6x10 6 cells/mL using the High Titer protocol. (Blue line) High density transfection at 10x10 6 cells/mL Figure 8. Ultra-High Density Transfection for Rapid Protein Expression Days post transfection hIgG Titer (g/L) 0 1 2 3 4 5 6 7 0 1 2 10x10 6 /mL 6x10 6 /mL Days post transfection hIgG Titer (g/L) 0 1 2 3 4 5 6 7 0.0 0.5 1.0 1.5 10x10 6 /mL 6x10 6 /mL Expi293 Expi293 Standard Protocol High Titer Protocol VIII. ExpiCHO System Scalability (A) ExpiCHO is directly scalable from 125mL to 2L flask sizes, 3L flasks require reduction in shake speed to 70rpm. (B) High Titer protocol in 24 deep well plates with shake speed of 225rpm. Figure 9. Scalability of the ExpiCHO System A B IX. Expression of Select Proteins in ExpiCHO System X. Protein Characterization Expression levels of human IgG, Rabbit IgG, Murine IgG and a broad range of non-Ab proteins in Expi293 or other transient expression systems and ExpiCHO system are shown. ExpiCHO IgG titers range from 0.6-97x (Avg. 5x) those obtained using various other systems. Additionally, 6 proteins from this panel were non- expressors in 293 cells yet expressed at high levels in ExpiCHO. Table 1. Titer Comparison in Expi293 or Current and ExpiCHO systems Std High Max Stable Expi293 Std High Max Stable Expi293 Reduced purified IgG Non-Reduced purified IgG ExpiCHO ExpiCHO A ExpiCHO Standard Protocol ExpiCHO High Titer Protocol ExpiCHO Max Titer Protocol 86% 92% 91% 6.069 7.279 7.659 8.853 10.597 AU 0.000 0.010 0.020 0.030 0.040 0.050 Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 6.117 7.247 7.664 8.855 10.640 11.965 AU 0.000 0.010 0.020 0.030 0.040 0.050 Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 82% 2% 1% Stable CHO-S Expi293 95% 92% B C ExpiCHO Stable CHO-S Expi293 87% 7% 3% 58% 20% 9% (A) Human IgG SDS PAGE (Left) Non-Reduced and (Right) Reduced purified IgG. (B) Human IgG SEC. (C) Human IgG Glycans. Figure 10. Protein Quality and Glycosylation Patterns in ExpiCHO and Expi293 A B Protein Expi293 or Current System (mg/L) ExpiCHO (mg/L) Fold Increase Protein Expi293 or Current System (mg/L) ExpiCHO (mg/L) Fold Increase Protein Expi293 or Current System (mg/L) ExpiCHO (mg/L) Fold Increase Human Ab 26.6 2580 97 Human Ab 352 371 1.1 Bi-TE Western Blot Western Blot 25 4 147 33 565 620 1.1 Bi-Specific 28 117 4.2 4 56 14.6 309 302 1.0 26 90 3.5 22 266 12.4 139 129 0.9 126 250 2.0 48 334 6.9 11 9 0.8 NA 763 36 190 5.3 565 365 0.6 NA 660 18 78 4.3 Undetectable 500 Secreted 216 1412 6.5 85 314 3.7 Precipitated 330 Non-Ab 15 82 5.5 170 410 2.4 Rabbit Ab 5 120 20 Western Blot Western Blot 5.0 138 325 2.4 17 178 11 164 68 0.4 25 50 2.0 Murine Ab 216 1412 6.5 NA 206 102 204 2.0 20 86 4.3 Fab 85 366 4.3 57 92 1.6 60 240 4.0 10 10 1.0 336 491 1.5 60 240 4.0 FC-Fusion 5 8 1.7 565 864 1.5 76 196 2.6 His Tag HLA Undetectable 12 12 450 600 1.3 12 18 1.5 Viral protein Western Blot Western Blot 3-5 350 450 1.3 16 17 1.1 Cytokine Western Blot Western Blot 5-10 309 383 1.2 119 48 0.4 FcR Western Blot Western Blot 2-3 243 276 1.1 135 47 0.3
Transcript of ExpiCHO™: Surpassing the Performance of 293 in a Transient ... · PDF fileability to...
PEI Max ExpiFectamineCHO 0
1
2
3
hIgG
Tite
r (g
/L)
(No Enhancer or Feed) (With Enhancer and Feed)
6.113
7.240
7.637
8.850
10.621
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
6.816 7.232
7.633
8.848
10.651
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
6.110
7.450 7.635
8.854
10.663
11.963
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Days of Culture
hIgG
Tite
r (g/
L)
0 1 2 3 4 5 6 7 8 9 10 110.0
0.5
1.0
1.5
2.0
Control 125 mL Flask High Titer Protocol
Chao Yan Liu, Virginia Spencer, Shyam Kumar, Jian Liu, Ping Liu, Sara Barnes, Henry Chiou*, Jonathan F. Zmuda. Thermo Fisher Scientific, 7335 Executive Way, Frederick MD, *5791 Van Allen Way, Carlsbad, CA.
(A) ExpiCHO-S cells. (B) Stability of protein expression over 18 passages. (C) Growth curve for ExpiCHO-S cells grown in standard shake flask culture.
Figure 6. ExpiCHO Protocol(s)
ABSTRACT & INTRODUCTION CHO cells are the predominant host for biotherapeutic protein expression, with roughly 70% of licensed biologics manufactured in CHO. Multiple attributes make CHO cells desirable for bioproduction including the ability to adapt to high-density suspension culture in serum-free and chemically-defined media and the incorporation of post-translational modifications that are biologically-active in humans. For these reasons, the ability to produce transient CHO-derived proteins early on during drug development is highly advantageous to minimize, as much as possible, changes in protein quality/function observed when moving from R&D to bioproduction. Unfortunately, CHO cells express lower levels of protein than HEK293 cells in existing transient systems, in some instances 50-100 times less than the best 293-based systems, and only modest titer improvements are obtained through the optimization of individual components of existing transient CHO workflows. To address the significant unmet need for higher transient CHO protein titers, systems-based approaches were employed whereby the latest advances in cell culture media, feeds, transfection reagents and expression enhancers were optimized in conjunction with a new high-expressing CHO cell clone to generate a simple and robust workflow for transient protein expression in CHO cells capable of generating gram per liter protein titers in 10-14 days. These advances will allow for unprecedented access to CHO-derived proteins early on during candidate selection and may serve to revolutionize the use of CHO cells for transient protein expression during the drug development process.
CONCLUSIONS We describe a systems-based approach for enhancing levels of transient protein production in CHO cells that allows for the production of recombinant proteins at levels exceeding those of the Expi293 system while maintaining activity, purity and glycosylation patterns comparable to those observed in stably transfected CHO-S cells. This performance enhancement was made possible through the incorporation of multiple novel reagents including: (1) a high-expressing CHO cell clone, (2) a CD/AOF culture media that allows for high density CHO growth and transfection, (3) an optimized CHO cell transfection reagent, (4) a novel CHO feed optimized for transient transfection culture conditions, (5) a post-transfection enhancer solution and (6) a simple to perform workflow.
ACKNOWLEDGEMENTS We would like to thank Michael Gillmeister for performing the glycan analysis on the human IgG samples and Brian Paszkiet for assisting with the transfection efficiency studies.
ExpiCHO™: Surpassing the Performance of 293 in a Transient CHO Expression System
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
Figure 1. Systems-Based Approach to Increased Transient Protein Expression
Figure 2. Workflow for Identifying High-Expressing CHO Clones
CHO cells were transiently transfected and evaluated for protein expression using the ClonePix system (Molecular Devices). Selected clones were further evaluated via transfection with plasmids for multiple proteins. Control protein titers using an existing CHO clone as well as Control titers for the pre-clonal pool.
ExpiCHO-S Cell Line Attributes • Derived from GMP CHO-S cells • Adapted for high-density culture • High specific productivity (~27pcd) • No clumping during expression runs • Short doubling time (~17 hours) • Stable growth and expression over 20+passages
A
B C
Figure 3. Characterization of ExpiCHO-S Cells
V. Workflow
ExpiCHO Expression Media Attributes • No supplementation required • One medium for growth and transfection • Formulated specifically for transient transfection • Chemically-defined (CD) • Animal origin-free (AOF) • Serum-free • Protein-free • Manufactured under cGMP • Supports high-density cell growth • Matched to a specific feed • Free from regulatory/import/export limitations
III. ExpiCHO Expression Medium and ExpiCHO Feed
hIgG
Tite
r (g
/L)
ExpiCHO Enhancer
0
1
2
3
4
_ +
hIgG
Tite
r (g
/L)
One Two 0
1
2
3
ExpiCHO Feed
(A) Protein titers are reduced by 50% or more without the addition of the ExpiCHO Enhancer reagent. (B) Two equal volume feeds on Days 1 and 5 post-transfection double protein titers.
Figure 4. Optimization of ExpiCHO Enhancer and Feed Addition
IV. ExpiFectamineCHO Transfection Reagent
(A) When used in conjunction with ExpiCHO Feed and Enhancer, ExpiFectamineCHO generates greater than 30-fold higher titers than PEI alone. (B) Despite the high density of cells at the time of transfection, plasmid DNA levels as low as 0.5 µg/mL of culture volume generate maximal protein titers, corresponding to half of the industry standard of 1.0 µg/mL plasmid DNA.
Figure 5. Characteristics of ExpiFectamineCHO Transfection Reagent
0.2 0.4 0.6 0.8 1.0 1.20
1
2
3
hIgG
Tite
r (g
/L)
DNA Concentration (µg/mL)
30 fold
II. ExpiCHO Expression Medium and ExpiCHO Feed Viability Viable Cell Density Titer
VI. Kinetics of Protein Production A B C
A B
(A) (Green line) Standard Protocol consisting of one feed and no temperature shift. (Blue line) High Titer Protocol consisting of one feed and temperature shift to 32ºC. (Red line) hIgG kinetics using the Max Titer Protocol consisting of two feeds and temperature shift to 32ºC. (B) Viability post transfection. (C) Viable cell density post transfection for Standard and Max Titer protocols.
Figure 7. Kinetics of hIgG Expression, Viability and Viable Cell Density
VII. System Flexibility: Ultra-High Density Transfection
Ultra-high density transfection can generate protein titers in half the time. (A) (Red line) Transfection of ExpiCHO-S cells at density of 6x106 cells/mL using the Standard protocol. (Blue line) High density transfection of ExpiCHO-S cells at 10x106 cells/mL. (B) (Red line) Transfection of ExpiCHO cells at density of 6x106 cells/mL using the High Titer protocol. (Blue line) High density transfection at 10x106 cells/mL
Figure 8. Ultra-High Density Transfection for Rapid Protein Expression Days post transfection
hIgG
Tite
r (g
/L)
0 1 2 3 4 5 6 70
1
210x106/mL
6x106/mL
Days post transfection
hIgG
Tite
r (g
/L)
0 1 2 3 4 5 6 70.0
0.5
1.0
1.5
10x106/mL
6x106/mL
Expi293 Expi293
Standard Protocol High Titer Protocol
VIII. ExpiCHO System Scalability
(A) ExpiCHO is directly scalable from 125mL to 2L flask sizes, 3L flasks require reduction in shake speed to 70rpm. (B) High Titer protocol in 24 deep well plates with shake speed of 225rpm.
Figure 9. Scalability of the ExpiCHO System
A B
IX. Expression of Select Proteins in ExpiCHO System
X. Protein Characterization
Expression levels of human IgG, Rabbit IgG, Murine IgG and a broad range of non-Ab proteins in Expi293 or other transient expression systems and ExpiCHO system are shown. ExpiCHO IgG titers range from 0.6-97x (Avg. 5x) those obtained using various other systems. Additionally, 6 proteins from this panel were non-expressors in 293 cells yet expressed at high levels in ExpiCHO.
Table 1. Titer Comparison in Expi293 or Current and ExpiCHO systems
Std High Max Stable Expi293 Std High Max Stable Expi293
Reduced purified IgG Non-Reduced purified IgG
ExpiCHO ExpiCHO
A
ExpiCHO Standard Protocol
ExpiCHO High Titer Protocol
ExpiCHO Max Titer Protocol
86% 92% 91%
6.069
7.279
7.659
8.853
10.597
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
6.117 7.247
7.664
8.855
10.640
11.965
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
82%
2% 1%
Stable CHO-S Expi293 95% 92%
B
C ExpiCHO Stable CHO-S Expi293
87% 7% 3%
58% 20% 9%
(A) Human IgG SDS PAGE (Left) Non-Reduced and (Right) Reduced purified IgG. (B) Human IgG SEC. (C) Human IgG Glycans.
Figure 10. Protein Quality and Glycosylation Patterns in ExpiCHO and Expi293
A B
Protein
Expi293 or Current System (mg/L)
ExpiCHO (mg/L)
Fold Increase
ProteinExpi293 or Current
System (mg/L)
ExpiCHO (mg/L)
Fold Increase
ProteinExpi293 or Current
System (mg/L)
ExpiCHO (mg/L)
Fold Increase
Human Ab 26.6 2580 97 Human Ab 352 371 1.1 Bi-TE Western Blot Western Blot 254 147 33 565 620 1.1 Bi-Specific 28 117 4.24 56 14.6 309 302 1.0 26 90 3.522 266 12.4 139 129 0.9 126 250 2.048 334 6.9 11 9 0.8 NA 76336 190 5.3 565 365 0.6 NA 66018 78 4.3 Undetectable 500 Secreted 216 1412 6.585 314 3.7 Precipitated 330 Non-Ab 15 82 5.5
170 410 2.4 Rabbit Ab 5 120 20 Western Blot Western Blot 5.0138 325 2.4 17 178 11 164 68 0.425 50 2.0 Murine Ab 216 1412 6.5 NA 206
102 204 2.0 20 86 4.3 Fab 85 366 4.357 92 1.6 60 240 4.0 10 10 1.0
336 491 1.5 60 240 4.0 FC-Fusion 5 8 1.7565 864 1.5 76 196 2.6 His Tag HLA Undetectable 12 12450 600 1.3 12 18 1.5 Viral protein Western Blot Western Blot 3-5350 450 1.3 16 17 1.1 Cytokine Western Blot Western Blot 5-10309 383 1.2 119 48 0.4 FcR Western Blot Western Blot 2-3243 276 1.1 135 47 0.3
